Cloning and sequence analysis of the nmnat gene
Based on the amino acid similarity to Dichomitus squalens nmnat (NCBI Reference Sequence: XP_007369986.1), only the gene Glnmnat (GenBank Accession Number: MH394247) was screened from the G. lucidum genome database (http://www.herbalgenomics.org/galu/). The length of the nmnat cDNA was 891 bp, and predictions indicated that this cDNA could encode a protein of 297 amino acids with a molecular mass of 32.89 kDa and an isoelectric point of 6.05. Analysis of the predicted amino acid sequence of Glnmnat identified the two highly conserved ATP-binding domains SxTxxR motif and GxxxPx[T/H]xxH motif, which can be easily detected in multiple sequence alignments and are commonly found in the nmnat-like proteins  (Fig. 1). These results implied that the Glnmnat gene that was cloned could belong to the nmnat family.
Construction of G. lucidum nmnat overexpression strains and measurement of NAD+ content
To explore the function of NAD+ in G. lucidum, the OE::nmnat vector was constructed (Fig. S1A), and Agrobacterium tumefaciens-mediated transformation (ATMT) was performed to construct OEnmnatmutant strains. The PCR results showed that the putative transformants presented the hygromycin B phosphotransferase (hph) gene and the gpd promoter-nmnat gene (Fig. S1B). Quantitative reverse-transcription PCR (qRT-PCR) analysis was utilized to detect the transcription level of nmnat in the selected mutant strains. Thirty positive transformants were selected, and seven of them had a higher transcript levels, with the relative expression of nmnat approximately 3–6-fold higher than the WT strains (Fig. S1C). OE::nmnat4 and OE::nmnat19 strains were randomly selected for further analysis. (Fig. 2A). The nmnat gene is a key gene in the synthesis of NAD+; therefore, the NAD+ content in the two nmnat overexpression strains was measured. The NAD+ content in G. lucidum was increased 1.35-fold and 1.38-fold in the OE::nmnat4 and OE::nmnat19 strains, respectively, compared with the WT strains (Fig. 2B). This result was consistent with the trend of nmnat gene expression at the transcript level. The increase in the NAD+ content further indicated that the cloned gene was a functional nmnat gene.
G. lucidum nmnat overexpression strains displayed better growth characteristics
Early studies have shown that the content of NAD+ might play an important role in response to nutritional conditions . To investigate the influence of NAD+ on the mycelial growth of G. lucidum on different carbon sources, all tested strains were inoculated on MM (minimal medium) agar plates with 1% carbon sources, namely, glucose, sucrose, lactose, glycerol, xylan, CMC-Na and lignin. Glnmnat overexpression strains showed dramatically stronger colony growth on these carbon sources than WT strains (Fig. 3A and B). The results implied that NAD+ might be involved in the process of utilizing nutrition.
Effects of NAD+ on cellulase production ofG. lucidum
To further investigate the role of NAD+ in nutrient utilization in G. lucidum, the same weight of precultured mycelia of G. lucidum in CYM liquid was transferred to liquid MM containing 1% Avicel as the sole carbon source and for the determination of cellulase activity. As shown in Fig. 4A and B, CMCase activity (representing endo-β-glucanase activity) in OE::nmnat4 and OE::nmnat19 strains approximately increased by 2.8-fold and 3-fold, respectively, compared with that of the WT strain. pNPGase activity (representing β-glucosidase activity) in OE::nmnat4 and OE::nmnat19 strains increased by approximately 1.9-fold and 2.1-fold compared with that of the WT strain, respectively. Meanwhlie, the total protein concentrations of all strains also was determined by a BCA Protein Assay Kit. As shown in Fig. 4C, there were no significant differences in total protein concentrations between WT strains and overexpression strains. The protein secretion is not directly correlated with the growth of the mycelium, and may be a result of complex inducing factors.
In addition, the level of transcription of the genes encoding major cellulases and transcription factors in G. lucidum was evaluated. Three putative endoglucanase coding genes (GL24196, GL29421, and GL28282), three putative cellobiohydrolase coding genes (GL18725, GL 29727, and GL30351) and three putative beta-glucosidase coding genes (GL27550, GL20743, and GL24911), respectively, were selected from the genome of G. lucidum. As shown in Fig. 4D, the gene expression of the major cellulases was significantly upregulated in the Glnmnat overexpression strains compared with that in the WT strains. Then, two negative transcription regulators for producing cellulase, creA (GL19424) and ace1 (GL15296), and two positive transcription regulators for producing cellulase, clr–1 (GL26482) and clr–2 (GL15667), were obtained. These results showed that the expression of clr–1 and clr–2 were significantly upregulated in the Glnmnat overexpression strains compared with the WT strains, while the expression of creA and ace1 were significantly downregulated in the Glnmnat overexpression strains compared with the WT strains (Fig. 4E). These results were consistent with the increasing of cellulase activity and implied that NAD+ might influence cellulase production in G. lucidum.
Effect of NAD+ on the accumulation of Ca2+ in G. lucidum
Previous studies have demonstrated that the calcium signal transduction pathway can upregulate cellulase gene expression . Therefore, Fluo–3-pentaacetoxymethyl ester (Fluo–3AM), an acetoxymethyl ester of a Ca2+-specifc probe, was used to detect relative amounts of free intracellular Ca2+. As shown in Fig. 5A and B, the fluorescence intensity of OE::nmnat4 and OE::nmnat19 strains was significantly higher than that of the WT strain, and the Ca2+ fluorescence value was upregulated by approximately 3.95-fold and 2.10-fold, respectively, compared with the WT strain. To investigate whether the increased levels of cytosolic Ca2+ can trigger calcium signal transduction pathways in G. lucidum, qRT-PCR was carried out to analyse the transcriptional levels of calcium signalling-related genes in G. lucidum, including calmodulin (cam),, Ca2+ and cam-dependent protein kinase genes (camk1, camk2 and camk3),, calreticulin [regulatory] gene, calcineurin [catalytic] genes (cna1 and cna2),, and calcineurin-responsive zinc finger transcription factor gene (crz).. As shown in Fig. 5C, the expression of most genes were increased to varying degrees in G. lucidum nmnat overexpression strains compared with the WT strain. These results implied that NAD+ might increase the concentration of cytosolic Ca2+, thus stimulating the calcium signal transduction pathway in G. lucidum.
NAD+ regulates cellulase activity via intracellular Ca2+
LaCl3, a plasma membrane Ca2+ channel blocker, was used to prevent influx of external Ca2+. As shown in Fig. 6A and B, the cytosolic Ca2+ concentration could be effectively attenuated in G. lucidum nmnat overexpression strains after the addition of LaCl3. Furthermore, CMCase and pNPGase activities and transcription of related genes were analysed. As expected, after adding 5 mM LaCl3 to G. lucidum nmnat overexpression mutants, both the CMCase and pNPGase activities decreased significantly (Fig. 7A and B), and the transcription of genes encoding the major cellulases also exhibited a similar tendency (Fig. 7C~K). Similarly, the transcription of clr–1 and clr–2 was markedly reduced in G. lucidum nmnat overexpression mutants with 5 mM LaCl3 treatment, while the transcription of creA and ace1 was markedly improved (Fig. 7L~O). Taken together, these data indicated that NAD+ might induce cellulase improvement via the Ca2+ concentration in G. lucidum.